Abstract

ABSTRACT
Cellular oxygen is sensed by prolyl-4-hydroxylase domain (PHD) proteins that
hydroxylate hypoxia-inducible factor (HIF) α subunits. Under normoxic conditions,
hydroxylated HIFα is bound by the von Hippel-Lindau (pVHL) tumor suppressor, leading to ubiquitinylation and proteasomal degradation. Under hypoxic conditions hydroxylation becomes reduced, leading to HIFα stabilization. We recently showed that changes in PHD abundance and activity can regulate HIFα stability under normoxic as well as under hypoxic conditions. Thus,
the PHD oxygen sensors themselves represent effectors of cellular signalling pathways as well as potential drug targets. Here, we applied a cell-free in vitro microtiter plate-based peptide hydroxylation assay to investigate the influence of ferrous iron, Krebs cycle intermediates, transition metals, vitamin C and other antioxidants on the activity of purified PHD1 to 3. PHD
activity depends not only on oxygen availability but is also regulated by iron, vitamin C and Krebs cycle intermediates, suggesting a physiological relevance of their cellular concentrations.
Copper but not iron, cobalt or nickel salts catalyzed vitamin C oxidation. While vitamin C is essential for PHD activity in vitro, N-acetyl-L-cysteine had no effect, and gallic acid or n-propyl gallate efficiently inhibited the activity of all three PHDs, demonstrating different functions of these antioxidants.

Abstract

ABSTRACT
Cellular oxygen is sensed by prolyl-4-hydroxylase domain (PHD) proteins that
hydroxylate hypoxia-inducible factor (HIF) α subunits. Under normoxic conditions,
hydroxylated HIFα is bound by the von Hippel-Lindau (pVHL) tumor suppressor, leading to ubiquitinylation and proteasomal degradation. Under hypoxic conditions hydroxylation becomes reduced, leading to HIFα stabilization. We recently showed that changes in PHD abundance and activity can regulate HIFα stability under normoxic as well as under hypoxic conditions. Thus,
the PHD oxygen sensors themselves represent effectors of cellular signalling pathways as well as potential drug targets. Here, we applied a cell-free in vitro microtiter plate-based peptide hydroxylation assay to investigate the influence of ferrous iron, Krebs cycle intermediates, transition metals, vitamin C and other antioxidants on the activity of purified PHD1 to 3. PHD
activity depends not only on oxygen availability but is also regulated by iron, vitamin C and Krebs cycle intermediates, suggesting a physiological relevance of their cellular concentrations.
Copper but not iron, cobalt or nickel salts catalyzed vitamin C oxidation. While vitamin C is essential for PHD activity in vitro, N-acetyl-L-cysteine had no effect, and gallic acid or n-propyl gallate efficiently inhibited the activity of all three PHDs, demonstrating different functions of these antioxidants.

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